Shoot meristem specific promoter sequences

ABSTRACT

A novel tissue-specific promoter is provided which has been isolated from the upstream non-coding region of a plant UFO gene. This promoter, operably associated with a nucleic acid sequence expressing a product of interest, initiates and regulates the transcription of such sequences in a shoot meristem-specific tissue.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No.IBN-9406948 awarded by the National Science Foundation. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to plant genetic engineering, andspecifically to a tissue specific promoter capable of directing shootmeristem-specific expression.

BACKGROUND OF THE INVENTION

Genes are regulated in an inducible, tissue specific or constitutivemanner. There are different types of structural elements which areinvolved in the regulation of gene expression. Cis-acting elements,located in the proximity of, or within genes, serve to bindsequence-specific DNA binding proteins, i.e., trans-acting factors. Thebinding of proteins to DNA is responsible for the initiation,maintenance, or down-regulation of gene transcription.

Cis-acting elements which control genes include promoters, enhancers andsilencers. Promoters are positioned next to the transcription start siteand function in an orientation-dependent manner, while enhancer andsilencer elements, which modulate the activity of promoters, may beflexible with respect to their orientation and distance from thetranscription start site.

Various promoter sequences are available which may be used in thegenetic engineering of plants. Such promoters may be utilized toinitiate transcription of a nucleic acid sequence of interest operablylinked at the 3' end of the promoter region. Promoters often havetranscription specific characteristics such as strength, tissuespecificity, developmental stage specificity, etc.

Gene expression in plants may be driven by a number of promoters.Although the endogenous promoter of a gene of interest may be utilizedfor transcriptional regulation of the gene, the promoter may also be aforeign regulatory sequence. Examples of viral promoters utilized inplant expression vectors include the 35S RNA and 19S RNA promoters ofCaMV (Brisson, et al., Nature, 310:511, 1984; Odell, et al., Nature,313:810, 1985); the full-length transcript promoter from Figwort MosaicVirs (FMV) (Gowda, et al., J. Cell Biochem., 13D: 301, 1989) and thecoat protein promoter of TMV (Takamatsu, et al., EMBO J. 6:307, 1987).Plant promoters also include the light-inducible promoter from the smallsubunit of ribulose bis-phosphate carboxylase (ssRUBISCO) (Coruzzi, etal., EMBO J., 3:1671, 1984; Broglie, et al., Science, 224:838, 1984);mannopine synthase promoter (Velten, et al., EMBO J., 3:2723, 1984)nopaline synthase (NOS) and octopine synthase (OCS) promoters (carriedon tumor-inducing plasmids of Agrobacterium tumefaciens) and heat shockpromoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley, et al., Mol.Cell. Biol., 6:559, 1986; Severin, et al., Plant Mol Biol., 15:827,1990).

Promoters utilized in plant genetic engineering include bothconstitutive and inducible natural promoters as well as engineeredpromoters. The CaMV promoters are examples of constitutive promoters. Tobe useful, an inducible promoter should 1) provide low expression in theabsence of the inducer; 2) provide high expression in the presence ofthe inducer; 3) use an induction scheme that does not interfere with thenormal physiology of the plant; and 4) have no effect on the expressionof other genes. Examples of inducible promoters useful in plants includethose induced by chemical means, such as the yeast metallothioneinpromoter which is activated by copper ions (Mett, et al., Proc. Natl.Acad. Sci., U.S.A., 90:4567, 1993); In2-1 and In2-2 regulator sequenceswhich are activated by substituted benzenesulfonamides, e.g., herbicidesafeners (Hershey, et al., Plant Mol. Biol., 17:679, 1991); and the GREregulatory sequences which are induced by glucocorticoids (Schena, etal., Proc. Natl. Acad. Sci., U.S.A., 88:10421, 1991).

Tissue specific promoters may also be utilized for expression of genesin plants. Tissue specific promoters useful in transgenic plants includethe cdc2a promoter and cyc07 promoter (Ito, et al., Plant Mol. Biol.,24:863, 1994; Martinez, et al., Proc. Natl. Acad. Sci. USA, 89:7360,1992; Medford, et al., Plant Cell, 3:359, 1991; Terada, et al., PlantJournal, 3:241, 1993; Wissenbach, et al., Plant Journal, 4:411, 1993).Additional tissue specific promoters that are utilized in plants includethe histone promoter (Atanassova, et al., Plant Journal, 2:291, 1992);the cinnamyl alcohol dehydrogenase (CAD) promoter (Feuillet, et al.,Plant Mol. Biol., 27:651, 1995); the mustard CHS1 promoter (Kaiser, etal., Plant Mol Biol., 28:231, 1995); the bean grp 1.8 promoter (Keller,et al., Plant Mol. Biol., 26:747, 1994); the PAL1 promoter (Ohl, et al.,Plant Cell, 2:837, 1990); and the chalcone synthase A promoter (PlantMol. Biol., 15:95-109, 1990).

SUMMARY OF THE INVENTION

The present invention provides a novel tissue-specific promoter isolatedfrom the Unusual Floral Organ gene (UFO). Transgenic plants, in whichthe invention promoter is fused to a nucleic acid sequence expressing aproduct of interest, exhibit phenotypes that indicate that the promotercan drive functional expression of a heterologous gene in shootmeristems.

In a first embodiment, the invention provides a nucleic acid constructcomprising a non-coding regulatory sequence isolated from a plantUnusual Floral Organs (UFO) gene and a nucleic acid sequence, whereinsaid nucleic acid sequence expresses a product selected from a proteinof interest or antisense RNA, and wherein said nucleic acid sequence isheterologous to the non-coding sequence. The construct is useful for theproduction of transgenic plants which express a gene of interest in ashoot meristem-specific manner.

In a second embodiment, the invention provides transgenic plant cellscomprising the nucleic acid constructs of the invention as well asplants comprising such cells.

In a further embodiment, the invention provides a method of providingincreased transcription of a nucleic acid sequence expressing a productselected from a protein of interest or antisense RNA. The methodcomprises providing a plant having integrated in its genome a nucleicacid construct of the invention and subjecting the plant to conditionssuitable for growth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1 is the nucleotide and deduced amino acid sequence of the UFOgene and some 5' and 3' noncoding sequences (SEQ IDNO:1 and 2,respectively).

FIG. 1A-2 is the nucleotide sequence of the 3'-terminal 2.6 kb of theUFO promoter. The initiation codon, ATG, is underlined (SEQ IDNO:3).

FIG. 2 is a comparison of UFO and FIM protein sequences and location ofufo mutations associated with alleles ufo-2 through ufo-6 (SEQ IDNO:4and5, respectively).

FIG. 3 is a restriction map of the UFO promoter.

FIG. 4(A-D) shows photographs of GUS activity in UFO::GUS transgenicplants during early heart stage (panel A), the torpedo stage (panel B),young seedlings (panel C), and after floral induction (panel D).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a novel promoter sequence that is usefulfor shoot meristem-specific gene expression. The shoot apical meristemforms the primary plant body and often undergoes differentiation to forma flower. In accordance with the present invention, a nucleic acidconstruct is provided which allows for modification of a plant phenotypebased on expression of a desired gene in the shoot meristem. The nucleicacid construct comprises a sequence of interest which provides for themodification in phenotype, positioned downstream from and under thetranscriptional initiation regulation of the invention shoot-specificpromoter. The shoot meristem-specific promoter is useful for specificexpression, in the shoot meristem, of genes involved in regulatingdevelopment. Such genes include those involved in flowering, as well asgenes that protect against pathogens by encoding toxins.

In a first embodiment, the invention provides a nucleic acid constructcomprising a non-coding regulatory sequence isolated upstream from aplant Unusual Floral Organs (UFO) gene, wherein the non-codingregulatory sequence is operably associated with a nucleic acid sequenceexpressing a product selected from a protein of interest or antisenseRNA and wherein said nucleic acid sequence is heterologous to saidnon-coding sequence. The construct includes a transcriptional andtranslational initiation region and a transcriptional and translationaltermination region functional in plants. In preparing the construct, thevarious component nucleic acid sequences may be manipulated, so as toprovide for nucleic acid sequences in the proper orientation and in theproper reading frame.

The UFO gene regulatory sequence, or promoter, is located in thenon-coding region of the gene and exhibits strong expression in theshoot apical meristem. Approximately 4 kilobases of 5' non-codingsequence was isolated upstream from the coding sequence, as described inthe Examples described herein. FIG. 1B shows the sequence ofapproximately 2.6 kb of the 3'-terminal region of the promoter. The maintranscription start site is at -225 bp relative to the ATG initiationcodon which is underlined. The transcription initiation sequencesinclude transcriptional control regions such as TATAA and CAAT boxsequences as well as sequences which regulate the tissue specificity ofthe transcribed product. In the nucleic acid construct of the invention,the ATG start codon is typically provided by the nucleic acid sequenceexpressing the product of interest.

One may identify a convenient restriction site in the 5'-untranslatedregion of the UFO gene and in the 5' region of the nucleic acid sequenceexpressing the product of interest and employ an adapter which will jointhe two sequences. Alternatively, one may introduce a polylinkerimmediately downstream from the UFO noncoding region for insertion ofthe nucleic acid sequence expressing the product of interest.

Placing a nucleic acid sequence expressing a product of interest underthe regulatory control of a promoter or a regulatory element meanspositioning the sequence such that expression is controlled by thepromoter or regulatory element. In general, promoters are positionedupstream of the genes that they control. Thus, in the construction ofpromoter/gene combinations, the promoter is preferably positionedupstream of the gene and at a distance from the transcription start sitethat approximates the distance between the promoter and the gene itcontrols in its natural setting. As is known in the art, some variationin this distance can be tolerated without loss of promoter function.Similarly, the preferred positioning of a regulatory element withrespect to a gene placed under its control reflects its natural positionrelative to the structural gene it naturally regulates. Again, as isknown in the art, some variation in this distance can be accommodated.The 5'-noncoding sequences which are used in the invention construct arenot more than about 4 kbp in length.

Promoter function during expression of a gene under its regulatorycontrol can be tested at the transcriptional stage using DNA/RNA andRNA/RNA hybridization assays (in situ hybridization) and at thetranslational stage using specific functional assays for the proteinsynthesized (for example, by enzymatic activity or by immunoassay of theprotein).

As used herein, the term "nucleic acid sequence" refers to a polymer ofdeoxyribonucleotides or ribonucleotides, in the form of a separatefragment or as a component of a larger construct. Nucleic acidsexpressing the products of interest can be assembled from cDNA fragmentsor from oligonucleotides which provide a synthetic gene which is capableof being expressed in a recombinant transcriptional unit. Polynucleotideor nucleic acid sequences of the invention include DNA, RNA and cDNAsequences.

Nucleic acid sequences utilized in the invention can be obtained byseveral methods. For example, the DNA can be isolated usinghybridization procedures which are well known in the art. These include,but are not limited to: 1) hybridization of probes to genomic or cDNAlibraries to detect shared nucleotide sequences; 2) antibody screeningof expression libraries to detect shared structural features and 3)synthesis by the polymerase chain reaction (PCR). Sequences for specificgenes can also be found in GenBank, National Institutes of Healthcomputer database.

The phrase "nucleic acid sequence expressing a product of interest"refers to a structural gene which expresses a product selected from aprotein of interest or antisense RNA. The term "structural gene"excludes the non-coding regulatory sequence which drives transcription.The structural gene may be derived in whole or in part from any sourceknown to the art, including a plant, a fungus, an animal, a bacterialgenome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNAor chemically synthesized DNA. A structural gene may contain one or moremodifications in either the coding or the untranslated regions whichcould affect the biological activity or the chemical structure of theexpression product, the rate of expression or the manner of expressioncontrol. Such modifications include, but are not limited to, mutations,insertions, deletions and substitutions of one or more nucleotides. Thestructural gene may constitute an uninterrupted coding sequence or itmay include one or more introns, bound by the appropriate splicejunctions. The structural gene may also encode a fusion protein. It iscontemplated that introduction into plant tissue of nucleic acidconstructs of the invention will include constructions wherein thestructural gene and its promoter e.g., UFO promoter, are each derivedfrom different plant species.

The term "heterologous nucleic acid sequence" as used herein refers toat least one structural gene which is operably associated with theregulatory sequence or promoter of the invention. The nucleic acidsequence originates in a foreign species, or, in the same species ifsubstantially modified from its original form. For example, the term"heterologous nucleic acid sequence" includes a nucleic acid originatingin the same species, where such sequence is operably linked to apromoter that differs from the natural or wild-type promoter (e.g., UFOpromoter).

The term "operably associated" refers to functional linkage between thepromoter sequence and the structural gene regulated by the promotersequence. The operably linked promoter controls the expression of theproduct expressed by the structural gene.

Examples of structural genes that may be employed in the presentinvention include the Bacillus thuringiensis toxin gene, which providespest/pathogen protection and the LEAFY gene, which controls flowering inplants. A variety of structural genes of interest that can be operablylinked to the promoter of the invention are available. For example,various sequences may be employed relating to enhanced resistance topesticides. Such sequences provide for the expression of a protein toxinderived from, for example, Bacillus thuringiensis. Moreover, sequencesrelating to herbicides may also be employed. Such sequences can providefor the expression of a mutated 5-enolpyruvyl-2-phosphoshikimatesynthase to provide decreased sensitivity to glyphosate. Such sequencescan also provide for the expression of a gene product involved indetoxification of bromoxynil. Sequences may also be employed relating toenhanced resistance to stress (such as provided by a gene for superoxidedismutase), temperature changes, osmotic pressure changes and salinity(such as a gene associated with the overproduction of proline) and thelike. Growth of the shoot meristem may be modulated, either increased ordecreased, depending on the particular need. Antisense sequences may beused to reduce growth or other phenotypic traits.

The term "genetic modification" or "genetically modified" as used hereinrefers to the introduction of one or more heterologous nucleic acidsequences into one or more plant cells, which can generate whole,sexually competent, viable plants. The term "genetically modified" asused herein refers to a plant which has been generated through theaforementioned process. Genetically modified plants of the invention arecapable of self-pollinating or cross-pollinating with other plants ofthe same species so that the foreign gene, carried in the germ line, canbe inserted into or bred into agriculturally useful plant varieties. Theterm "plant cell" as used herein refers to protoplasts, gamete producingcells, and cells which regenerate into whole plants. Plant cells includecells in plants as well as protoplasts in culture. Accordingly, a seedcomprising multiple plant cells capable of regenerating into a wholeplant, is included in the definition of "plant cell". Plant tissueincludes differentiated and undifferentiated tissue derived from roots,shoots, pollen, seeds, tumor tissue, such as crown galls, and variousforms of aggregations of plant cells in culture, such as embryos andcalluses.

As used herein, the term "plant" refers to either a whole plant, a plantpart, a plant cell, or a group of plant cells, such as plant tissue, forexample. Plantlets are also included within the meaning of "plant".Plants included in the invention are any plants amenable totransformation techniques, including angiosperms, gymnosperms,monocotyledons and dicotyledons.

Examples of monocotyledonous plants include, but are not limited to,asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion,pearl millet, rye and oats. Examples of dicotyledonous plants include,but are not limited to tomato, tobacco, cotton, rapeseed, field beans,soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops orBrassica oleracea (e.g., cabbage, broccoli, cauliflower, brusselsprouts), radish, carrot, beats, eggplant, spinach, cucumber, squash,melons, cantaloupe, sunflowers and various ornamentals. Woody speciesinclude poplar, pine, sequoia, cedar, oak, etc.

Genetically modified plants of the present invention are produced bycontacting a plant cell with an invention nucleic acid construct asdescribed above. The construct is preferably contained within a vector.Vector(s) employed in the present invention for transformation of aplant cell for shoot meristem expression comprise a nucleic acidsequence comprising at least one structural gene expressing a product ofinterest, operably associated with the promoter of the invention. It ispreferred that the vector harboring the heterologous nucleic acidsequence also contain one or more selectable marker genes so that thetransformed cells can be selected from non-transformed cells in culture,as described herein.

As used herein, the term "marker" refers to a gene encoding a trait or aphenotype which permits the selection of, or the screening for, a plantor plant cell containing the marker. Preferable, the marker gene is anantibiotic resistance gene whereby the appropriate antibiotic can beused to select for transformed cells from among cells that are nottransformed. Examples of suitable selectable markers include adenosinedeaminase, dihydrofolate reductase, hygromycin-beta-phosphotransferase,thymidine kinase, exanthineguanine phospho-ribosyltransferase andamino-glycoside 3'-O-phosphotransferase II. Other suitable markers willbe known to those of skill in the art.

To commence a transformation process in accordance with the presentinvention, it is first necessary to construct a suitable vector andproperly introduce it into the plant cell. The details of theconstruction of the vectors then utilized herein are known to thoseskilled in the art of plant genetic engineering.

For example, the nucleic acid sequences utilized in the presentinvention can be introduced into plant cells using Ti plasmids,root-inducing (Ri) plasmids, and plant virus vectors. (For reviews ofsuch techniques see, for example, Weissbach & Weissbach, 1988, Methodsfor Plant Molecular Biology, Academic Press, NY, Section VIII, pp.421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9, and Horsch, et al., Science, 227:1229, 1985,both incorporated herein by reference).

One of skill in the art will be able to select an appropriate vector forintroducing the invention nucleic acid construct in a relatively intactstate. Thus, any vector which will produce a plant carrying theintroduced nucleic acid construct should be sufficient. Even a nakedpiece of nucleic acid would be expected to be able to confer theproperties of this invention, though at low efficiency. The selection ofthe vector, or whether to use a vector, is typically guided by themethod of transformation selected.

The transformation of plants in accordance with the invention may becarried out in essentially any of the various ways known to thoseskilled in the art of plant molecular biology. (See, for example,Methods of Enzymology, Vol.153, 1987, Wu and Grossman, Eds., AcademicPress, incorporated herein by reference). As used herein, the term"transformation" means alteration of the genotype of a host plant by theintroduction of a nucleic acid construct as described herein.

For example, a construct of the invention can be introduced into a plantcell utilizing Agrobacterium tumefaciens containing the Ti plasmid. Inusing an A. tumefaciens culture as a transformation vehicle, it is mostadvantageous to use a non-oncogenic strain of the Agrobacterium as thevector carrier so that normal non-oncogenic differentiation of thetransformed tissues is possible. It is also preferred that theAgrobacterium harbor a binary Ti plasmid system. Such a binary systemcomprises 1) a first Ti plasmid having a virulence region essential forthe introduction of transfer DNA (T-DNA) into plants, and 2) a chimericplasmid. The chimeric plasmid contains at least one border region of theT-DNA region of a wild-type Ti plasmid flanking the nucleic acid to betransferred. Binary Ti plasmid systems have been shown effective totransform plant cells (De Framond, Biotechnology, 1:262, 1983; Hoekema,et al., Nature, 303:179, 1983). Such a binary system is preferredbecause it does not require integration into Ti plasmid inAgrobacterium.

Methods involving the use of Agrobacterium include, but are not limitedto: 1) co-cultivation of Agrobacterium with cultured isolatedprotoplasts; 2) transformation of plant cells or tissues withAgrobacterium; or 3) transformation of seeds, apices or meristems withAgrobacterium.

In addition, gene transfer can be accomplished by in situ transformationby Agrobacterium, as described by Bechtold, et al., (C.R. Acad. Sci.Paris, 316:1194, 1993) and exemplified in the Examples herein. Thisapproach is based on the vacuum infiltration of a suspension ofAgrobacterium cells.

The preferred method of introducing a nucleic acid construct of theinvention into plant cells is to infect such plant cells, an explant, ameristem or a seed, with transformed Agrobacterium tumefaciens asdescribed above. Under appropriate conditions known in the art, thetransformed plant cells are grown to form shoots, roots, and developfurther into plants.

Alternatively, a nucleic acid construct of the invention can beintroduced into a plant cell by contacting the plant cell usingmechanical or chemical means. For example, the nucleic acid can bemechanically transferred by microinjection directly into plant cells byuse of micropipettes. Alternatively, the nucleic acid may be transferredinto the plant cell by using polyethylene glycol which forms aprecipitation complex with genetic material that is taken up by thecell.

A nucleic acid construct of the invention can also be introduced intoplant cells by electroporation (Fromm, et al., Proc. Natl. Acad. Sci.,U.S.A, 82:5824, 1985, which is incorporated herein by reference). Inthis technique, plant protoplasts are electroporated in the presence ofvectors or nucleic acids containing the relevant nucleic acid sequences.Electrical impulses of high field strength reversibly permeabilizemembranes allowing the introduction of nucleic acids. Electroporatedplant protoplasts reform the cell wall, divide and form a plant callus.Selection of the transformed plant cells with the transformed gene canbe accomplished using phenotypic markers as described herein.

Another method for introducing nucleic acid into a plant cell is highvelocity ballistic penetration by small particles with the nucleic acidto be introduced contained either within the matrix of small beads orparticles, or on the surface thereof (Klein, et al., Nature 327:70,1987). Although, typically only a single introduction of a new nucleicacid sequence is required, this method particularly provides formultiple introductions.

Cauliflower mosaic virus (CaMV) may also be used as a vector forintroducing a nucleic acid construct of the invention into plant cells(U.S. Pat. No. 4,407,956). CaMV viral DNA genome is inserted into aparent bacterial plasmid creating a recombinant DNA molecule which canbe propagated in bacteria. After cloning, the recombinant plasmid againmay be cloned and further modified by introduction of the desirednucleic acid sequence. The modified viral portion of the recombinantplasmid is then excised from the parent bacterial plasmid, and used toinoculate the plant cells or plants.

In another embodiment, the invention affords a method of providingincreased transcription of a nucleic acid sequence expressing a productof interest in shoot meristem tissue. The method comprises providing aplant having integrated into its genome a nucleic acid construct of theinvention and subjecting the plant to conditions suitable for growthwhereby transcription of the nucleic acid sequence is increased in theshoot meristem tissue.

Typically, the nucleic acid construct is introduced into a plant cell bycontacting the cell with a vector containing the promoter-nucleic acidsequence encoding the protein of interest construct. As used herein, theterm "contacting" refers to any means of introducing the vector(s) intothe plant cell, including chemical and physical means as describedabove. Preferably, contacting refers to introducing the nucleic acid orvector into plant cells (including an explant, a meristem or a seed),via Agrobacterium tumefaciens transformed with the heterologous nucleicacid as described above.

Normally, a plant cell is regenerated to obtain a whole plant from thetransformation process. The immediate product of the transformation isreferred to as a "transgenote". The term "growing" or "regeneration" asused herein means growing a whole plant from a plant cell, a group ofplant cells, a plant part (including seeds), or a plant piece (e.g.,from a protoplast, callus, or tissue part).

Regeneration from protoplasts varies from species to species of plants,but generally a suspension of protoplasts is first made. In certainspecies, embryo formation can then be induced from the protoplastsuspension, to the stage of ripening and germination as natural embryos.The culture media will generally contain various amino acids andhormones, necessary for growth and regeneration. Examples of hormonesutilized include auxin and cytokinins. It is sometimes advantageous toadd glutamic acid and proline to the medium, especially for such speciesas corn and alfalfa. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. If these variablesare controlled, regeneration is reproducible.

Regeneration also occurs from plant callus, explants, organs or parts.Transformation can be performed in the context of organ or plant partregeneration (see Methods in Enzymology, Vol. 118 and Klee, et al.,Annual Review of Plant Physiology, 38:467, 1987). Utilizing the leafdisk-transformation-regeneration method of Horsch, et al., Science,227:1229, 1985, disks are cultured on selective media, followed by shootformation in about 2-4 weeks. Shoots that develop are excised from calliand transplanted to appropriate root-inducing selective medium. Rootedplantlets are transplanted to soil as soon as possible after rootsappear. The plantlets can be repotted as required, until reachingmaturity.

In vegetatively propagated crops, the mature transgenic plants arepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenotesis made and new varieties are obtained and propagated vegetatively forcommercial use.

In seed propagated crops, the mature transgenic plants can be selfcrossed to produce a homozygous inbred plant. The inbred plant producesseed containing the newly introduced foreign gene(s). These seeds can begrown to produce plants that would produce the selected phenotype, e.g.early flowering.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells that have been transformed asdescribed. Progeny and variants, and mutants of the regenerated plantsare also included within the scope of the invention, provided that theseparts comprise the introduced nucleic acid sequences.

The invention includes a plant produced by the method of the invention,including plant tissue, seeds, and other plant cells derived from thegenetically modified plant.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLES

Meristems in the aerial portion of a plant have to choose between twoalternative fates, shoot or flower meristem, a decision that isregulated by a set of meristem-identity genes (Weigel, D., Annu. Rev.Genetics, 29:19, 1995). Such genes include the snapdragon gene FIMBRIATA(FIM), whose inactivation causes a partial transformation of flowersinto shoots (Simon, et al., Cell, 78:99, 1994). The FIM gene has beencloned and shown to be specifically expressed in young flowers (Simon,et al., supra). A gene with significant sequence similarity to FIM hasbeen isolated from Arabidopsis thaliana and shown to correspond to theUFO gene, mutations in which cause phenotypes similar to that ofmutations in FIM (Ingraham, et al., Plant Cell, 7:1501, 1995; Levin andMeyerowitz, Plant Cell, 7:529, 1995; Wilkinson an Haughn, Plant Cell,7:1485, 1995).

The present invention describes isolation of the UFO coding andnoncoding nucleic acid sequences. UFO RNA was found to be expressed notonly in flowers as previously reported (Ingram, et al., supra), but alsoin shoot meristems. To test whether the UFO promoter is sufficient todrive expression of a heterologous gene, the promoter was fused to thestructural gene β-glucuronidase (GUS). Transgenic Arabidopsis andtobacco plants that carry a fusion of the UFO promoter to a reportergene encoding β-glucuronidase (GUS) were constructed. These plantsexpress high levels of GUS in shoot meristems, as determined byhistochemical staining with the GUS substrate X-gluc(5-bromo-4-chloro-3-indoyl β-D-glucuronide). Functional activity of theUFO promoter in shoot meristems was confirmed by generating transgenicArabidopsis plants in which the UFO promoter is fused to the nucleicacid sequence encoding the protein of interest encoding the LEAFY (LFY)gene product. High levels of LFY expression are normally restricted toyoung flowers (Weigel, et al., Cell, 69:843, 1992), and transgenicplants in which LFY is constitutively expressed show transformation ofshoots into flowers, apparently due to ecotopic expression of LFY inshoot meristems (Weigel and Nilsson, Nature, 377:495, 1995). A similarphenotype is observed in UFO::LFY plants, indicating that the UFOpromoter can drive functional expression of a heterologous gene in shootmeristems.

Example 1 Isolation and Identification of the UFO Gene

The UFO gene was isolated using probes for the snapdragon FIM gene.Genomic DNA was extracted from locally purchased snapdragons, and twoadjacent portions of the FIM coding region were amplified by polymerasechain (PCR) reaction (Saiki, et al., Science, 239:487, 1988; Simon, etal., supra). The PCR products were radioactively labeled and used toscreen a lambda vector library of Arabidopsis genomic DNA. Duplicatefilters were hybridized with the non-overlapping FIM probes, and clonesthat hybridized to both probes were purified (Sambrook, et al.,Molecular Cloning, 2nd Edition, Cold Spring Harbor: Cold Spring HarborLaboratory, 1989). The FIM cross-hybridizing region was subcloned intoplasmid vectors (pBluescript, Stratagene) and the DNA sequence wasdetermined by the Sanger method (Sambrook, et al., supra.) (FIG. 1A).Analysis of the genomic sequence identified an uninterrupted openreading frame of 1326 base pairs, with coding potential for a 442 aminoacid long protein that shares significant similarity with the FIMprotein (FIG. 2).

To confirm that this gene corresponded to UFO, a CAPS marker (Koniecznyand Ausubel, Plant J., 4:403, 1993) was developed and the map positionof the gene determined by mapping it against a previously characterizedset of recombinant inbred lines (Lister and Dean, Plant J., 4:745,1993). The derived map position at 68 cM on chromosome I agrees with thegenetic linkage of ufo mutations to the CAL locus on chromosome I (Levinand Meyerowitz, supra). Genomic DNA was extracted from five ufo mutantalleles, ufo-2 through ufo-6 (Levin and Meyerowitz, supra), and the UFOcoding region was amplified by PCR (Saiki, et al., supra). Sequencingrevealed single amino acid changes in all five alleles (FIG. 2). Thefour strong alleles ufo-2 through ufo-5 are all associated with nonsensemutations predicted to cause a truncation of the UFO protein. The weakallele ufo-6 has a missense mutation. The map position together with themutant sequences showed that this clone was derived from the UFO gene.Further confirmation was obtained by complementation of the ufo-2mutations with a transgene in which the UFO coding region is under thecontrol of the 35S promoter from cauliflower mosaic virus. This promoteris described in Odell, et al., Nature, 313:810, 1985. These resultsagree with the results reported by Ingraham, et al., (1995 codingregion; GenBank database, accession number X89224).

Example 2 Analysis of UFO Expression and Promoter Sequences

Using in situ hybridization to sections of Arabidopsis plants, the UFOgene was found to be expressed in shoot meristems as well as in youngflowers. To study the activity of the UFO promoter, approximately 4 kbof upstream sequences were fused to the GUS reporter (Jefferson, et al.,supra). In brief, a BamHI restriction site was introduced downstream ofthe initiation codon, to produce the sequence ATGGATcC (initiation codonindicated in bold, mutated nucleotide indicated by lower case letter).The 5' fragment used extends from an EcoRI site to this artificial BamHIsite (FIG. 3). The fragment was cloned as an HindIII/BamHI fragmentupstream of the GUS coding region in the backbone of the pCGN1547 T-DNAtransformation vector (McBride and Summerfelt, Plant Mol. Biol., 14:269,1990), yielding pDW228, which was transformed into Agrobacteriumtumefaciens strain, ASE (Fraley, et al. Biotechnology, 3:629, 1985), andintroduced in to Arabidopsis thaliana ecotype Columbia plants by thevacuum infiltration method (Bechtold, et al., C.R. Acad. Sci., 316:1194,1993). Transgenic plants were selected on kanamycin, seeds wereharvested, and progeny were analyzed for GUS activity using the X-glucsubstrate at various times during development (Jefferson, et al.,EMBOJ., 6:3901, 1987).

Strong expression of GUS was first detected in the apical part of theembryo during early heart stage (FIG. 4, panel A). As the shoot apicalmeristems forms during the torpedo stage, GUS activity becomesrestricted to the shoot apical meristem (FIG. 4, panel B). In youngseedlings that are not florally induced, the same pattern was detected(FIG. 4, panel C). Once floral induction has occurred, GUS activity isdetected in the shoot apical meristem and in young flowers (FIG. 4,panel D). GUS activity is also detected in axillary shoot meristems thatform in the axils of rosette leaves.

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 5    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3139 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 571..1900    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TTTATATTTCTGTTAGAAATAACAACATATATCAACTGATTTTTTACTTCCAATCTCTTT60    TTGTCAGCACACAAATAGAAAAACGTCTGTAAGCTAAGCTATCAACTAAAACATTAACAT120    ATATAATCTTTTACGTTGATAGAAAATAAACATAAATTTCTGAGTTATTTTTTTTTTGGT180    TGGTGTGTCACTACTTACTTACTACTATACCTTTTTAACAATAAAGAAACACTATTTCTT240    TTTCTATTCAATATAATATATGTTTTCTATTTGTATAAATCCATTACCTTTGTTTGTTTT300    ATACCAAATGTTCTTTATATATAGTATATGCGACGTTACTCTATTGAAGTCAAGAACATA360    TCAAAAACCCATGCAAGAAGCTCACAGAGAAAGACGAAACGCTTTTGTCTCTTTCTTCAA420    AACTTTTACATATGATCTTTGCCTCTTTTCCTACAATGGGTTTTGCATAACTTTCACCAA480    AACCCTCCTCAAAAGCCCTTCACATATTCCCAACACAAGAAAATAAACTCTAAATCCACT540    TTCACCAAATCTTTTCATTTTTCAGCTAAAATGGATTCAACTGTGTTCATCAAT594    MetAspSerThrValPheIleAsn    15    AACCCATCTTTAACCTTACCTTTCTCTTACACATTTACCAGTAGCAGC642    AsnProSerLeuThrLeuProPheSerTyrThrPheThrSerSerSer    101520    AACAGTAGCACAACAACGAGCACCACCACAGACTCAAGCTCCGGTCAA690    AsnSerSerThrThrThrSerThrThrThrAspSerSerSerGlyGln    25303540    TGGATGGACGGTCGGATTTGGAGCAAGCTACCACCTCCTCTTCTTGAC738    TrpMetAspGlyArgIleTrpSerLysLeuProProProLeuLeuAsp    455055    CGCGTCATTGCTTTTCTTCCACCTCCGGCGTTTTTCCGGACACGTTGC786    ArgValIleAlaPheLeuProProProAlaPhePheArgThrArgCys    606570    GTCTGCAAGAGATTCTACAGTCTACTTTTCTCCAACACCTTCCTCGAG834    ValCysLysArgPheTyrSerLeuLeuPheSerAsnThrPheLeuGlu    758085    ACATATCTACAACTACTTCCTCTCCGACACAACTGTTTCCTCTTCTTC882    ThrTyrLeuGlnLeuLeuProLeuArgHisAsnCysPheLeuPhePhe    9095100    AAACACAAAACCCTAAAGAGTTACATTTACAAGAGAGGAGGAACAAAC930    LysHisLysThrLeuLysSerTyrIleTyrLysArgGlyGlyThrAsn    105110115120    GATGATGATTCCAATAAAGCTGAAGGCTTTTTGTTTGATCCTAATGAG978    AspAspAspSerAsnLysAlaGluGlyPheLeuPheAspProAsnGlu    125130135    ATCCGATGGTACCGTCTCTCTTTTGCTTATATCCCTTCAGGGTTTTAT1026    IleArgTrpTyrArgLeuSerPheAlaTyrIleProSerGlyPheTyr    140145150    CCTTCAGGATCATCAGGAGGGTTAGTGAGTTGGGTCTCCGAAGAAGCT1074    ProSerGlySerSerGlyGlyLeuValSerTrpValSerGluGluAla    155160165    GGTCTTAAAACCATTCTCTTGTGTAACCCTCTTGTCGGATCCGTGAGT1122    GlyLeuLysThrIleLeuLeuCysAsnProLeuValGlySerValSer    170175180    CAGTTGCCACCAATATCAAGGCCAAGGCTTTTCCCTTCGATAGGTCTC1170    GlnLeuProProIleSerArgProArgLeuPheProSerIleGlyLeu    185190195200    TCGGTAACACCAACCTCTATTGATGTTACTGTCGCTGGAGATGATCTC1218    SerValThrProThrSerIleAspValThrValAlaGlyAspAspLeu    205210215    ATATCTCCTTACGCTGTGAAAAACCTCTCATCGGAGAGTTTCCATGTC1266    IleSerProTyrAlaValLysAsnLeuSerSerGluSerPheHisVal    220225230    GACGCCGGCGGATTCTTTTCCCTCTGGGCGATGACTTCTTCTTTGCCA1314    AspAlaGlyGlyPhePheSerLeuTrpAlaMetThrSerSerLeuPro    235240245    CGGCTTTGTAGCTTGGAATCTGGTAAGATGGTTTACGTGCAAGGCAAG1362    ArgLeuCysSerLeuGluSerGlyLysMetValTyrValGlnGlyLys    250255260    TTTTACTGTATGAACTATAGCCCTTTTAGCGTTTTGTCCTATGAAGTT1410    PheTyrCysMetAsnTyrSerProPheSerValLeuSerTyrGluVal    265270275280    ACTGGAAACCGGTGGATCAAGATTCAAGCTCCGATGAGGAGATTTCTC1458    ThrGlyAsnArgTrpIleLysIleGlnAlaProMetArgArgPheLeu    285290295    AGATCTCCAAGCTTGTTAGAGAGCAAAGGGAGGCTTATTCTTGTAGCA1506    ArgSerProSerLeuLeuGluSerLysGlyArgLeuIleLeuValAla    300305310    GCTGTTGAGAAAAGCAAGTTGAACGTTCCCAAAAGCCTACGACTTTGG1554    AlaValGluLysSerLysLeuAsnValProLysSerLeuArgLeuTrp    315320325    AGTTTGCAACAAGATAACGCCACATGGGTCGAGATCGAACGGATGCCT1602    SerLeuGlnGlnAspAsnAlaThrTrpValGluIleGluArgMetPro    330335340    CAGCCGCTCTACACACAGTTTGCAGCAGAAGAAGGTGGAAAAGGATTC1650    GlnProLeuTyrThrGlnPheAlaAlaGluGluGlyGlyLysGlyPhe    345350355360    GAGTGTGTCGGAAATCAAGAGTTTGTAATGATTGTGTTAAGAGGAACC1698    GluCysValGlyAsnGlnGluPheValMetIleValLeuArgGlyThr    365370375    TCGTTGCAGTTGCTGTTTGATATAGTGAGAAAAAGCTGGCTGTGGGTC1746    SerLeuGlnLeuLeuPheAspIleValArgLysSerTrpLeuTrpVal    380385390    CCACCGTGTCCTTACTCCGGCAGTGGTGGCGGTAGCTCAGGTGGCGGT1794    ProProCysProTyrSerGlySerGlyGlyGlySerSerGlyGlyGly    395400405    TCAGACGGAGAGGTCTTGCAGGGTTTTGCTTATGACCCGGTGCTTACT1842    SerAspGlyGluValLeuGlnGlyPheAlaTyrAspProValLeuThr    410415420    ACACCGGTGGTTAGTCTTCTTGATCAGTTAACACTTCCATTTCCTGGA1890    ThrProValValSerLeuLeuAspGlnLeuThrLeuProPheProGly    425430435440    GTCTGTTAGTTTTTAGACTTTAAGATAAAGAGACTACTTGTGGTTTCCAC1940    ValCys*    TTCTGACGTTAAGACTGCTTGTTGTTTTCTCAAAATTCTGTTTCTTTTATCTTATTACTG2000    TCTGTATGTAGTAAGTTTATATTTCTAATGTCAAATGTCTAATCTTTGACAACATGTCAA2060    CAACATATACAGACAGATTTCTAATTGCGTACAATCCAATCCAATCCTAAATCCATCAAA2120    CTCAAAAACATAACCCTTGGGAGAATGGTTTCACTTGAGCTTAACCTGGAGAATGAGATG2180    AACTTTTCTGTTCATTATTCTCCTGAGTTCTTCATTGGCCTCAATTCCTATCCTCCTGCA2240    AAATTAGCATCAACATAAGATCATCCTTGAGTCATTGATTAGTCAAAAGAATGAATTATG2300    ACCGATCTTGTATGCTCTTACCCGATCTTGCAACCGCCCTTGCCTACAAGAATCTTGCGT2360    TGGCTAAGTTTAGGAGTGATGAGATGCTGTTCAATTCTAAGAGACCCGTCACGCAGCTCT2420    TTCCAGTCCACTAGACGGTGCTCCAGACCATATGGTATTTCCTACAATTTGTTTCAATCA2480    ATCCTGTAATTTGTCATCTTGGGATAGACGGAAACTGATACAAAATGTTATACTAGTAGA2540    GGTTGTATTATTACCTGATGGACATGGTCTAGTAATCTCTCCCTAACAACTTCAAGAGAA2600    ATGTTCTTCAAGACTTCTTCACTCATCGTGAATGCATCTTCTTCCCATGGTTTTTTAACA2660    GCCTGATCCATTAAGTATTGGGAAAGATCTTTCACTCCTGATCCCTTAAGTCCCGATATC2720    ATGAAGTATCTAGAAAACCAAACAGGGAAAAAGCACATTTCAATCAATTCGAAGACTTCC2780    CCGGTAATTGTTTTTAAACTGAGTCTGGTATATATATATCCACCTTTCATATGCCGGAAG2840    ATCTTGGAACTCCTCAGCAACCTTTAATAGATCCTTTTTCTTCTCAACCAGATCAACTTT2900    GTTCATACATAAAACGCGCTTTTGTTTCGGATTTTCTTCTTCTCCCATGTATTTGATCAA2960    GCGTACCACTCTTGAATCGGGACTAGATAAATCAAAGAGAAAACAGAATTCACAAACTAC3020    AAAATAAGTCTAAGAGAACTCTATTTCTACTTGTAATTAATGAAAAACCACAGTCATGAT3080    GCCTTCTTCAAAAGAAAGAAATAGATGTGTCTTCCCATCGGTTTACGGTTCTCAAGCTT3139    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 442 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAspSerThrValPheIleAsnAsnProSerLeuThrLeuProPhe    151015    SerTyrThrPheThrSerSerSerAsnSerSerThrThrThrSerThr    202530    ThrThrAspSerSerSerGlyGlnTrpMetAspGlyArgIleTrpSer    354045    LysLeuProProProLeuLeuAspArgValIleAlaPheLeuProPro    505560    ProAlaPhePheArgThrArgCysValCysLysArgPheTyrSerLeu    65707580    LeuPheSerAsnThrPheLeuGluThrTyrLeuGlnLeuLeuProLeu    859095    ArgHisAsnCysPheLeuPhePheLysHisLysThrLeuLysSerTyr    100105110    IleTyrLysArgGlyGlyThrAsnAspAspAspSerAsnLysAlaGlu    115120125    GlyPheLeuPheAspProAsnGluIleArgTrpTyrArgLeuSerPhe    130135140    AlaTyrIleProSerGlyPheTyrProSerGlySerSerGlyGlyLeu    145150155160    ValSerTrpValSerGluGluAlaGlyLeuLysThrIleLeuLeuCys    165170175    AsnProLeuValGlySerValSerGlnLeuProProIleSerArgPro    180185190    ArgLeuPheProSerIleGlyLeuSerValThrProThrSerIleAsp    195200205    ValThrValAlaGlyAspAspLeuIleSerProTyrAlaValLysAsn    210215220    LeuSerSerGluSerPheHisValAspAlaGlyGlyPhePheSerLeu    225230235240    TrpAlaMetThrSerSerLeuProArgLeuCysSerLeuGluSerGly    245250255    LysMetValTyrValGlnGlyLysPheTyrCysMetAsnTyrSerPro    260265270    PheSerValLeuSerTyrGluValThrGlyAsnArgTrpIleLysIle    275280285    GlnAlaProMetArgArgPheLeuArgSerProSerLeuLeuGluSer    290295300    LysGlyArgLeuIleLeuValAlaAlaValGluLysSerLysLeuAsn    305310315320    ValProLysSerLeuArgLeuTrpSerLeuGlnGlnAspAsnAlaThr    325330335    TrpValGluIleGluArgMetProGlnProLeuTyrThrGlnPheAla    340345350    AlaGluGluGlyGlyLysGlyPheGluCysValGlyAsnGlnGluPhe    355360365    ValMetIleValLeuArgGlyThrSerLeuGlnLeuLeuPheAspIle    370375380    ValArgLysSerTrpLeuTrpValProProCysProTyrSerGlySer    385390395400    GlyGlyGlySerSerGlyGlyGlySerAspGlyGluValLeuGlnGly    405410415    PheAlaTyrAspProValLeuThrThrProValValSerLeuLeuAsp    420425430    GlnLeuThrLeuProPheProGlyValCys    435440    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2555 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CGAATCAGTTAATGTGGGCGACAAAATAAAATCAGATTATGTTTTATTATTAAGTTTAGA60    AAGAGATGCTTCATTAAAAGTTTATGCTTTAATGTTAAATCAGACTATTTATTATGGGTA120    CGACTTGTAAGCGTAATTCAAAGTTAAACAAACCTAGTTGGGAAATGGGAAGATAGATGA180    TTTGATAAAAACAAGACACTGTTTGGTTTCAGAGACTTCCTTTAGCATCAAAACACAAAC240    AAAAGCGAAGCCTCTTGAGTTACTGCAAATAGAGAATATTATTACCCTTTTGCGACTTGT300    CAGCTTCAGATATTCTCACTTGTATTATTATTTTCACGGTAAACAATGCCTTAAATAAGA360    AACCCTGATTGGACTTTTGATCTGACTCTACTCTTCACTCTTTCTTCTTCTTTATTTTCA420    GTCATGATGTCTCTCTAACCCTAATCTCAAAAAATCCAAACTCCTTTTATTTATTTCTAA480    ACCTTGATTATAGCTAGCAATGATTAATATAAGAATTTTTTTTCTGGATAAAGAATTAAT540    TAGAAATTGAGTTGTAAATGTTTTGTGATGTCTAAAATTCTTTTGTTTGCAAATTAGATG600    TAAATTGATATATTTGAGATTTGTATGAAAGCTAGTTTTATTTTCCCTAAACAAGAAGTT660    CATTATCTTGGACTTGGAGGTTTTAGAGTTTGAAAGAGTTTACAATTTATAAGAAAAAAT720    AATCACTATATATATATATATAGTGTATATAATGAATTGTTTCACATTAAATTGCAACAA780    CATCAAATAAGGGTAACATACTAACATATAGTTTGTTTGTTTACTCTTTAAAAAAAGGGG840    ATAAACTAAGAGGCTATTTTCTGCTATAATTTAGGAACAAACTGGATCACATGACAAAAA900    TGCATCATAATTCATATTAAATTTTGTGTATATCTATTTCTCATGTTTAGAAATAACATT960    CTTGTGTGTTATACATGTTATCAGTTTTTCTTCCTAGATGGAAGTTTTATTGTTGGAGTC1020    TTTTAAAACCATACTCACTATGTTCCTCTTTATTTGATGTTTTGGGATTTTAGATAGGAA1080    ATTAATAAAAAATATGTTTTCTATTTTTATAAAATTATTTTTTTGTTAGTTTAGTAATTT1140    TATTTTTCTTTTTTTATTATTAGTCACAAGCAAAAATAATAACAATATTTTTATAAAACA1200    TTAATTTTGGTCGAACAAGTAAAAATAACTCAAAACATCAAATAATTAGAAACTAAAAAA1260    GTAGATATTGTCAAATTTTGTGTTGAGTTCGAATAAGATAATGTGGTCTCCTCCAACAAA1320    ATTATTTAGAATAAATGCACTTCTATGACATTAGAGAACCAACTAATTTATTTAGAATAA1380    AACACACATATATATTAACATATAAAGTAATTCTAATTGGCTTGATATAATATATAAGTA1440    AAAAAATGATCTTAATAATCTCTAGTTTTCTTGGGTTGATCTCCACGAGTACAATTTGAC1500    TGACCATTATAGAAGTTGAGAAGCGTGCATGTAATAAAAGTTGTATATTACAAATTAGAG1560    AGGAAAAAGAAAGAAAGAAAAAAGATTTGAAGATGTGATCAAGTGTGAAAAGTATTGGAG1620    TAGTCTCCAAATTAATAATTTCGATGCTGGGCATTGACAAGATAACTCTGAAGCTCTCAA1680    CTTTAAGACCATCACTTCCTCTCCACCATTTTCACGTTTACCCAAACACACACATATACA1740    AACAAAATTTTGTTAGTCAATAATTATCACCAAACTGGGGTTATAACAAGGCTTTTGGAT1800    ACTTGTGCTTGTTGATGTTCTAGGTTCGTATGATAACAAAGTACATCCGTTATATATATT1860    CGAAACACACTTTAATATTAAAAATATATATCCAATTTTCTTGTGAAATTTAGATTATTT1920    GGAATTAAACCTATTTCTCTTGTCTTGGCCACTTGACCGGTTTAGTTTTTTAGACGTATT1980    TTATTATTTCTGTTTAGAAAATAACAACATATATCAACTGATTTTTTACTTCCAATCTCT2040    TTTTGTCAGCACACAAATAGAAAAACGTCTGTAAGCTAAGCTATCAACTAAAACATTAAC2100    ATATATAATCTTTTACGTTGATAGAAAATAAACATAAATTTCTGAGTTATTTTTTTTTTG2160    GTTGGTGTGTCACTACTTACTTACTACTATACCTTTTTAACAATAAAGAAACACTATTTC2220    TTTTTCTATTCAATATAATATATGTTTTCTATTTGTATAAATCCATTACCTTTGTTTGTT2280    TTATACCAAATGTTCTTTATATATAGTATATGCGACGTTACTCTATTGAAGTCAAGAACA2340    TATCAAAAACCCATGCAAGAAGCTCACAGAGAAAGACGAAACGCTTTTGTCTCTTTCTTC2400    AAAACTTTTACATATGATCTTTGCCTCTTTTCCTACAATGGGTTTTGCATAACTTTCACC2460    AAAACCCTCCTCAAAAGCCCTTCACATATTCCCAACACAAGAAAATAAACTCTAAATCCA2520    CTTTCACCAAATCTTTTCATTTTTCAGCTAAAATG2555    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 442 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetAspSerThrValPheIleAsnAsnProSerLeuThrLeuProPhe    151015    SerTyrThrPheThrSerSerSerAsnSerSerThrThrThrSerThr    202530    ThrThrAspSerSerSerGlyGlnTrpMetAspGlyArgIleTrpSer    354045    LysLeuProProProLeuLeuAspArgValIleAlaPheLeuProPro    505560    ProAlaPhePheArgThrArgCysValCysLysArgPheTyrSerLeu    65707580    LeuPheSerAsnThrPheLeuGluThrTyrLeuGlnLeuLeuProLeu    859095    ArgHisAsnCysPheLeuPhePheLysHisLysThrLeuLysSerTyr    100105110    IleTyrLysArgGlyGlyThrAsnAspAspAspSerAsnLysAlaGlu    115120125    GlyPheLeuPheAspProAsnGluIleArgTrpTyrArgLeuSerPhe    130135140    AlaTyrIleProSerGlyPheTyrProSerGlySerSerGlyGlyLeu    145150155160    ValSerTrpValSerGluGluAlaGlyLeuLysThrIleLeuLeuCys    165170175    AsnProLeuValGlySerValSerGlnLeuProProIleSerArgPro    180185190    ArgLeuPheProSerIleGlyLeuSerValThrProThrSerIleAsp    195200205    ValThrValAlaGlyAspAspLeuIleSerProTyrAlaValLysAsn    210215220    LeuSerSerGluSerPheHisValAspAlaGlyGlyPhePheSerLeu    225230235240    TrpAlaMetThrSerSerLeuProArgLeuCysSerLeuGluSerGly    245250255    LysMetValTyrValGlnGlyLysPheTyrCysMetAsnTyrSerPro    260265270    PheSerValLeuSerTyrGluValThrGlyAsnArgTrpIleLysIle    275280285    GlnAlaProMetArgArgPheLeuArgSerProSerLeuLeuGluSer    290295300    LysGlyArgLeuIleLeuValAlaAlaValGluLysSerLysLeuAsn    305310315320    ValProLysSerLeuArgLeuTrpSerLeuGlnGlnAspAsnAlaThr    325330335    TrpValGluIleGluArgMetProGlnProLeuTyrThrGlnPheAla    340345350    AlaGluGluGlyGlyLysGlyPheGluCysValGlyAsnGlnGluPhe    355360365    ValMetIleValLeuArgGlyThrSerLeuGlnLeuLeuPheAspIle    370375380    ValArgLysSerTrpLeuTrpValProProCysProTyrSerGlySer    385390395400    GlyGlyGlySerSerGlyGlyGlySerAspGlyGluValLeuGlnGly    405410415    PheAlaTyrAspProValLeuThrThrProValValSerLeuLeuAsp    420425430    GlnLeuThrLeuProPheProGlyValCys    435440    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 164 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GluAlaPheGlnThrIlePheAsnLeuProGlyThrThrProThrIle    151015    AsnLeuGlnAsnMetIleMetThrThrAsnCysArgGlnLysIleIle    202530    CysSerSerTrpIleThrLeuHisAlaSerIleTrpMetGlnGlnSer    354045    IleHisHisAsnAsnAsnSerAlaArgProThrTyrTyrGlnThrLeu    505560    LysIleProLeuProSerAlaSerLeuIleCysAspSerProAsnSer    65707580    ThrAsnThrAlaIleSerThrLeuGluCysThrThrIleAsnSerIle    859095    SerPheThrIleValTyrIleAsnThrArgHisArgAspIleSerLeu    100105110    GlnCysThrValLysAlaGluCysGlyThrIleGlnIleGluIleArg    115120125    SerAlaHisAlaValLeuIleSerTyrAspLysAlaValMetPheCys    130135140    GlnValValAspAspGluHisGluArgAlaValIleThrProGluGln    145150155160    SerPheThrAla    __________________________________________________________________________

What is claimed is:
 1. A nucleic acid construct comprising a non-codingregulatory sequence isolated upstream from a plant Unusual Floral Organs(UFO) gene, wherein said non-coding regulatory sequence is operablyassociated with a nucleic acid sequence which expresses a protein ofinterest or antisense RNA, wherein said nucleic acid sequence isheterologous to said non-coding sequence, and wherein the non-codingregulatory region comprises a sequence as set forth in SEQ ID NO:3. 2.The nucleic acid construct of claim 1, wherein said non-coding sequencecomprises a transcriptional and translational initiation region.
 3. Thenucleic acid construct of claim 2, further comprising a transcriptionaltermination region functional in a plant cell.
 4. The nucleic acidconstruct of claim 1, wherein said nucleic acid sequence encodes aBacillus thuringiensis toxin.
 5. A transgenic plant cell comprising anucleic acid construct according to claim
 1. 6. The transgenic plantcell of claim 5, further comprising a selectable marker.
 7. A plantcomprising a transgenic plant cell according to claim
 5. 8. An isolatednucleic acid sequence comprising a non-coding regulatory sequenceisolated upstream from a plant Unusual Floral Organs (UFO) gene, whereinsaid nucleic acid sequence contains at least one restriction site forcloning a heterologous nucleic acid sequence of interest.
 9. A method ofproviding increased transcription of a nucleic acid sequence in shootmeristem tissue of a plant, said method comprising:subjecting agenetically engineered plant to conditions suitable for growth whereinsaid plant has integrated in its genome a nucleic acid constructcomprising a non-coding regulatory sequence isolated upstream from anUnusual Floral Organs (UFO) gene comprising a sequence as set forth inSEQ ID NO:3, wherein said non-coding regulatory sequence is operablyassociated with a nucleic acid sequence expressing a protein of interestor antisense RNA and wherein said nucleic acid sequence is heterologousto said non-coding sequence, whereby transcription of said nucleic acidsequence is increased in said meristem tissue.
 10. The method of claim9, wherein said non-coding region comprises a transcriptional andtranslational initiation region.
 11. The method of claim 9, wherein thenucleic acid sequence encodes a Bacillus thuringiensis toxin.
 12. Anisolated nucleic acid sequence comprising a nucleotide sequence as setforth in SEQ ID NO:3, wherein said nucleic acid sequence contains atleast one restriction site for cloning a heterologous nucleic acidsequence of interest.